Prostate cancer (CaP) is the most common malignancy and the second leading cause of cancer deaths in American men. The current clinical methods used for the detection of CaP are the serum prostate specific antigen (PSA) test, and the digital rectal examination (DRE). The PSA test was introduced into clinical practice two decades ago and has led to the detection of CaP at a potentially curable stage. Despite the high sensitivity of the PSA test (about 94%), a significant limitation is the very low specificity (about 20%), which is due to the fact that PSA is not a cancer-specific marker [1]. As a result, the clinical use of the PSA test has sparked controversy over the increased incidence in CaP observed in the U.S., which has led to the “over-diagnosis” and “overtreatment” of CaP [2]. A PSA level greater than/or equal to 4.0 ng/ml represents a clinical decision limit that prompts diagnostic biopsy testing [2]. However, a subset of patients with levels below 4.0 ng/ml may have or will develop CaP, and a large portion (65-75%) with greater than 4.0 ng/ml may have a noncancerous prostate-related disorder [3,4]. To increase the detection sensitivity of CaP, the PSA test is used along with the DRE; however, even when used together, the specificity of the screening procedure remains low, leading to unnecessary diagnostic biopsies (65-75% of all biopsies). The prostate biopsy, which can be painful, stressful and lead to infection, is the primary method used for the diagnostic confirmation of CaP [5].
Ideally a diagnostic biomarker should be detectable through noninvasive routes such as the collection and analysis of bodily fluids, including urine; and by methods that increase both sensitivity and specificity. Prostatic material can be acquired by the shedding of cells into the urine following prostate massage during a DRE [6]. Recently, a growing number of reports have demonstrated the benefit of using post-DRE urine in the diagnosis of CaP by the quantitative analysis of intracellular molecular markers rather than by whole cell visualization [4,7-12]. Other reported approaches for the detection of CaP in urine are cytology-based exploratory methods, such as ThinPre® (Hologic, Bedford, Mass.) filtration [14] or centrifugation methods, such as Cytospin® (Thermo Electron Corporation, Waltham, Mass.) methods [7], [13]. These methods have low sensitivity, which methods cannot reliably detect cancer cells in biological samples containing fewer than about 100 cells.
In centrifugation methods, such as Cytospin® (Thermo Electron Corporation, Waltham, Mass.) methods, centrifugal force is used to isolate and deposit cells on microscope slides. However, many cells can be lost during the centrifugation steps in these methods. Also, the centrifugal force can damage cells or cause them to lose their morphology, thereby preventing their subsequent detection. These methods also require a centrifuge and a unique sample chamber assembly comprising a microscope slide. As reported by Fujita et al., using the Cytospin® (Thermo Electron Corporation. Waltham, Mass.) method, LNCaP cells were detected only 50% of the time when urine samples were spiked with 100 LNCaP cells and no cells were detected when the urine samples were spiked with only 10 LNCaP cells.
ThinPrep® (Hologic, Bedford, Mass.) is a method that was first developed for the preparation of cervical cytology samples but has also been used for FISH analysis of bladder carcinoma in voided urine samples [14]. In the ThinPrep® (Hologic, Bedford, Mass.) method, cells are collected on a filter and then transferred to a microscope slide. Because the filter is not translucent, the cells cannot be detected directly on the filter but rather must be transferred to a microscope slide by gently pressing the filter against the slide. The ThinPrep® (Hologic, Bedford, Mass.) method is very effective for detecting cancer cells in biological samples containing a large number of cells but is not effective in detecting cancer cells in biological samples containing a limited number of cancer cells, particularly samples having fewer than about 100 cancer cells.
While shown to be beneficial in the detection of other cancers, these urinary cytology methods lack the sensitivity needed for the detection of biological samples containing very few cells of interest.
Another group has recently described a method for detecting circulating tumor cells using a proprietary, parylene filter [19]. The parylene filter is adapted to detect circulating tumor cells in blood, which are primarily tumor cells derived from metastasis. As such, this method is not designed to detect early stage cancer before metastatic progression. In addition, this method uses a formaldehyde-based reagent for fixing the cells in the biological sample, finding that the use of alcohol-based fixatives results in the formation of large aggregates of serum protein that quickly clog the filter and lead to failure of the device. Formaldehyde-based fixatives are more hazardous than alcohol-based fixatives, and formaldehyde can cause substantial autofluorescence, which interferes with the immunofluorescent detection and characterization of cells.
Therefore, other methods are needed to provide a sufficiently sensitive and specific method for the non-invasive detection of cancers in biological samples that contain very low concentrations of cancerous cells.